1. Field of the Invention
This invention relates to the field of medical diagnostic kits and a method for activity measurement of a protein. More particularly, it relates to a reagent device or kit that measures cholesteryl ester transfer protein (CETP) activity.
2. Description of the Prior Art
Cholesteryl ester transfer protein (CETP) is a protein that transfers cholesteryl ester (CE) from high-density lipoprotein (HDL) to low-density lipoprotein (LDL) and very-low density lipoprotein (VLDL). CETP will also transfer triglyceride (TG) among lipoprotein particles. For example, when a sample of VLDL or LDL, 1 or 10 micrograms of protein, respectively, is mixed with a sample of HDL, at total HDL cholesteryl ester of 4EE-10 moles, incubated at thirty seven degrees centigrade with a source of CETP, such as, one microliter of human plasma, cholesteryl ester will be transferred from the HDL to the LDL or VLDL particles. Typically, measurement of the CETP activity requires the cholesteryl ester associated with the HDL be provided with some type of label for monitoring the movement of the HDL cholesteryl ester to the LDL and VLDL components after incubation. The activity measurement techniques also usually require a final separation step after incubation so that either accumulation of HDL cholesteryl ester in LDL or VLDL may be quantified, or loss of HDL cholesteryl ester from HDL may be quantified. The HDL particle represents a donor of CE and the VLDL or LDL represent acceptors of CE.
There are several known techniques to measure cholesteryl ester transfer protein (CETP) activity. For example, an article entitled: Effect of Very Low-Density Lipoproteins on Lipid Transfer in Incubated Serum, by A. V. Nichols and L. Smith, J. Lipid Research, vol. 6, pp. 206-210 (1965), measures the activity of CETP by determination of cholesteryl ester (CE) mass transfer. The determination of CE mass transfer from high density lipoprotein (HDL) to very-low density lipoprotein (VLDL) and low density lipoprotein (LDL) requires the re-isolation of VLDL and LDL after incubation with HDL and the CETP source in order to determine the cholesteryl ester mass transfer.
The VLDL/LDL re-isolation from the incubation mixture is a technique that includes ultra-centrifugation of the incubation mixture for many hours so that the VLDL and LDL components are floated upwards through a density gradient as the HDL component of the incubation mixture sinks to the bottom of the centrifuge tube. Further processing of the sample requires a method of determining the amount or mass of cholesteryl ester associated with the re-isolated VLDL or LDL and equating a change in mass to CETP facilitated transfer. Later variations of this method of activity measurement have simplified mass determination by utilizing HDL that has a radioactive label associated with the CE.
While not stated in this article, the determination of CETP activity through tritium (3H) labeled cholesteryl ester (3H-CE) still requires the time consuming step of VLDL/LDL component re-isolation, or separation of VLDL or LDL from the 3H-CE containing HDL before the counts per minute of 3H-CE transferred can be determined. The present invention does not require the separation of any components of the incubation mixture nor does the present invention use radioactive isotopes.
An article entitled: Cholesteryl Ester Exchange Protein in Human Plasma Isolation and Characterization by N. M. Pattnaik, A. Montes, L. B. Hughes and D. B. Zilversmit, Biochimica et Biophysica Acta 530, pp. 428-438 (1978), discloses a method of activity measurement of CETP that also utilizes radioactive CE in HDL. This method is an improvement over the above method by simplifying the incubation mixture components separation or the re-isolation technique discussed above. In the cited article, separation of the LDL component from the HDL component is accomplished by precipitation of the LDL component of the incubation mixture. The LDL precipitate is pelleted by a relatively short, slow-speed centrifugation and the remaining HDL supernatant is counted. The loss of radioactivity from the HDL component is attributed to 3H-CE transferred to the LDL pellet. This method requires the use of radioactive isotopes and it is believed both prior art publications yield poor sensitivity and accuracy, characteristic of methods that require a high incident of sample manipulation.
An article titled: Fluorescent Determination of Cholesteryl Ester Transfer Protein (CETP) Activity in Plasma by N. Dousset and L. Douste-Blazy in Clinical Chemistry, vol. 38, No. 2, p. 306 (1982), is an improvement over previous methods of activity measurement since it discloses a technique that does not require radioactive components. In this method, transfer activity of the CETP is determined by the measurement of transfer of a fluorescent labeled CE. In this article, the cholesteryl ester molecule utilized as the CETP substrate for transfer, has been bonded to a fluorescent molecule derived from pyrene. The pyrene labeled cholesteryl ester (PY-CE) is recognized by the CETP and the PY-CE may be detected by a fluorimeter. The accumulation of the PY-CE in the LDL fraction is, however, only able to be determined after the separation of the LDL acceptor from the HDL donor.
An article entitled: Enhancement of The Human Plasma Lipid Transfer Protein Reaction by Apolipoproteins by T. G. Milner, K. W. S. Ko, T. Ohnishi and S. Yokoyama in Biochimica Biophysica Acta 1082, pp. 71-78 (1991), discloses a method for determining the activity of CETP also utilizing a pyrene labeled CE (PY-CE). This method does not require separation or re-isolation of substrates, but uses the measurement of both monomer and excimer fluorescent emission from the pyrene label to determine a ratio thereof. The cited article is improving upon certain aspects of the previous method. However, the method is based upon excimer to monomer ratio to determine accumulation of PY-CE in the acceptor and does not account for lipoprotein core viscosity changes affecting the excimer to monomer ratio. Pyrene labels have been used extensively in physical biochemistry to study particle core viscosity. This cited method results in problems with accuracy as noted in this article. The method is inconvenient due to oxygen quenching of excimer emission and requires the constant gassing of samples with nitrogen.
The present diagnostic kit is readily usable for the purpose of performing simple rapid and accurate tests to determine activity of CETP in a patient or group of patients. The kit does not utilize radioactive isotopes nor does the kit require the separation of donor and acceptor particles to accomplish activity measurements. The present kit yields a real time activity. Also, the reagent substrate emission is not subject to quenching by oxygen.